Study On The Effect And Mechanism Of Non-bone Metastatic Gastric Cancer On Osteoblast Differentiation

Part 1: Whether non-metastatic gastric cancer produces inflammatory factor CRP and the effect of CRP on the proliferation and osteoblast differentiation of OPCsThe progression of tumors is not only related to the internal characteristics of tumor cells,but also inseparable from the body’s inflammatory response.Generally,C-reactive protein(CRP)is considered to be an important factor in response to tissue infection and injury,and its concentration is a reliable sign of systemic inflammation.Its rapid increase is related to tumor necrosis factor alpha(TNF-α),interleukin 6(IL-6)and other pro-inflammatory cytokines,which accelerate the progression of many cancers,including gastric cancer.As we all know,bone metastasis is a serious complication in the later stage of cancer.It is reported that there is also a close correlation between CRP levels and bone mineral density,non-vertebral fractures and X-ray vertebral fractures.Therefore,this article will study the relationship between non-metastatic gastric cancer and CRP and the effect of CRP on the proliferation and differentiation of primary cultured SD rat neonatal skull osteoblast precursor cells(OPCs).We analyzed the serum calcium(Ca2+),phosphorus(Pi),alkaline phosphatase (ALP),high-sensitivity C-reactive protein(hs-CRP)and procalcitonin(PCT)in 25 patients with non-bone metastatic gastric cancer.Concentration and bone CT film,found that in the above patients,the serum Ca2+,Pi concentration dropped significantly(p<0.01),the concentration of ALP fluctuated with the normal value(p<0.05),the concentration of hs-CRP(p<0.001)and PCT(P<0.05)are mostly increased.Compared with normal people,3 bone CT films of gastric cancer patients showed obvious round and low-density shadows.In addition,BALB/c nude mice were injected with human gastric cancer cell line HGC-27 cells(1×107 cells/m L).After 90 days,the concentrations of Ca2+,Pi,ALP and CRP in the serum of the above two groups of nude mice were detected.Nude mice were sacrificed to collect femurs,and the degree of bone tissue damage was detected by H&E staining,micro-computed tomography(Micro-CT),immunohistochemistry,immunofluorescence and other techniques.The results of in vivo experiments showed that compared with BALB/c nude mice injected with PBS,the serum concentrations of Pi,Ca2+and ALP in BALB/c nude mice injected with HGC-27 cells significantly decreased(p<0.05),and the CRP concentration increased with time(p<0.01).H&E staining showed no gastric cancer bone metastasis,but the femur had obvious vacuoles and bone loss.Micro-CT results showed that the bone cancellous and bone compact in the HGC-27 cell injection group were significantly lower than the normal group,and the bone tolerance was also significantly reduced(p<0.05).Immunohistochemistry showed that the OPN positive area was significantly reduced compared with the control group(P<0.001).In addition,OPCs cells were randomly divided into the control group and the recombinant CRP treatment group,and the proliferation activity of the cells in each group was detected by CCK8 and Ki67 immunofluorescence.The results of in vitro experiments showed that, compared with the control group,the cell proliferation activity of OPCs in the recombinant CRP treatment group was significantly reduced(p<0.001).The effects of recombinant CRP on the cell cycle of OPCs were detected by cyclin D1 and cyclin D1 immunofluorescence,Western-blot and cell flow cytometry.The above results collectively indicated that recombinant CRP inhibited cell proliferation by blocking the G1/S transition period of OPCs.The effect of recombinant CRP on osteoblast differentiation of OPCs was detected by Alizarin Red staining,Western-blot,immunofluorescence and ALP activity.The above results together indicated that recombinant CRP inhibited the osteoblast differentiation of OPCs.Bioinformatics analysis,immunofluorescence and Western-blot were used to detect the effect of recombinant CRP on the primary cilia of OPCs.Bioinformatics showed that the primary cilia were important organelles involved in the process of osteogenic differentiation,and they abnormally activated the formation of primary cilia during the differentiation process of OPCs.By using CH(Chloral hydrate)to treat the OPCs cells in the recombinant CRP treatment group,the expression of OPCs cells’ cilia was significantly inhibited and was almost invisible(p<0.001).Western-blot was used to measure the expression of collagen 1and osteopontin(OPN)in the control group,the recombinant CRP treatment group and the chloral hydrate treatment recombinant CRP group.It was found that the addition of chloral hydrate rescued the loss of osteoblast differentiation of OPCs caused by the addition of recombinant CRP(p<0.001).These data indicate that the inflammatory factor CRP exerts its inhibitory effect on the proliferation and osteoblast differentiation of osteoblasts through cell primary cilia,suggesting that inhibiting cilia or blocking the release of inflammatory factors may be able to treat bone loss diseases caused by inflammation.Part 2: Study on whether non-metastatic gastric cancer can lead to bone loss and the molecular mechanismCancer-related bone disease is a high-risk complication in the later stages of cancer,accompanied by pain,bone fragility,bone loss,and fractures.However,whether primary or non-bone metastatic gastric cancer will induce bone loss remains unclear.Here,we injected BALB/c nude mice with human gastric cancer cell line HGC-27 cells(1x107 cells/m L).After 90 days,the nude mice were sacrificed and the femurs were collected,and the expression of primary cilia were detected by immunofluorescence.The results of in vivo experiments showed that the fluorescence intensity of acetylated α-tubulin and γ-tubulin in the bone tissue of the treatment group were significantly higher than that in the normal group,indicating that after stimulation by gastric cancer cells,the primary cilia of the bone tissue became longer and more(p<0.05).Immunofluorescence co-staining ofβ-catenin and acetylated α-tubulin in normal BALB/c nude mice showed that the expression sites of the two proteins were highly overlapped.After injection of HGC-27 cells,the expression of β-catenin in the bone tissue of nude mice was significantly increased than that of normal bone tissue(p<0.001),and the protein expression of phosphorylated β-catenin was significantly lower than that of normal bone tissue(p<0.001).In addition,MSC cells were randomly divided into non-co-culture group and HGC-27co-culture group,and the proliferation activity of cells in each group were detected by plate cloning,Ki67 and PCNA immunofluorescence.The results of in vitro experiments showed that the proliferation activity of MSCs co-cultured with HGC-27 cells was significantly decreased compared with non-co-cultured MSC cells(p<0.001).The HGC-27 cells were co-cultured with three types of osteogenic precursor cells(MC3T3-E1,MSCs and OPCs)in osteogenic induction medium for 16 days and then stained with Alizarin Red.The results showed that no matter which osteogenic precursor cells are associated with HGC-27 co-culture,there were almost no calcium ion deposition,while normal osteoblast precursor cells showed significant calcium ion deposition(p<0.001).Western-blot detected the osteogenic differentiation marker proteins ALP and OPN of MSCs co-cultured with SGC-7901,and it was found that the protein expression levels above the co-culture group were significantly decreased(p<0.001).The q RT-PCR test also got the same conclusion(p<0.05).The OCN protein immunofluorescence experiment was performed on the control group and the treatment group,which also confirmed the appeal result(p<0.001).Then we found that the fluorescence intensity of β-catenin and Wnt3 a in MSCs co-cultured with HGC-27 cells increased significantly compared with the control group(p<0.01).At the same time,we observed that β-catenin accumulated in the cytoplasm and gradually transferred to the nucleus.Next,we added the GES-1(normal gastric epithelial cell line)+ MSCs cell co-culture group,and the CH-treated HGC-27 cell + MSCs cell co-culture group were subjected to immunofluorescence staining to analyze the expression of primary cilia.The results showed that the immunofluorescence intensity of acetylated α-tubulin and γ-tubulin of MSCs co-cultured with HGC-27 cells were higher than that of MSCs(p<0.001),and the primary cilia became longer and more.In the GES-1 +MSCs cell co-culture group,there were almost no significant change in the primary cilia of MSCs.In the CH-treated HGC-27 + MSCs cell co-culture group,the expression of MSCs cell primary cilia were obviously suppressed and almost invisible(p<0.001).Western-blot detection of acetylated α-tubulin and γ-tubulin expression also reached the same conclusion(p<0.01).For the expression of related signal pathways,Naked1,Axin1 and β-catenin proteins were measured by Western-blot,and it was found that β-catenin of MSCs cells in the co-culture group of HGC-27 +MSC cells increased significantly(p<0.01),but Naked1 and Axin1 were significantly reduced(p<0.01).After CH treatment,the expression ofβ-catenin protein was significantly reduced(p<0.001),and there was no statistical difference in other proteins.Compared with the co-culture group of HGC-27 + MSC cells treated with DKK1 and the group of co-cultured HGC-27 + MSC cells without DKK1 treatment,the fluorescence intensity of immunofluorescence staining of acetylated α-tubulin and γ-tubulin,the length and number of primary cilia were not statistically significant differences.Western-blot detection of acetylated α-tubulin and γ-tubulin protein expression also yielded the same conclusion.After adding DKK1,the enrichment of β-catenin in the nucleus was significantly reduced,and the fluorescence intensity of Wnt3 a was also significantly weakened(p<0.05).Western-blot measured the protein expression of Naked1,Axin1 and β-catenin,and found that DKK1 rescued the effect of HGC-27 cells on MSCs.q RT-PCR found that after HGC-27 co-cultured,the m RNA transcription levels of Wnt3 a and β-catenin in MSCs cells were higher than those in control group(p<0.05),but the m RNA transcription level of TCF1 and GSK-3β were significantly lower(p<0.05).DKK1 rescued the effect of HGC-27 on MSCs cells(p<0.05).Three groups of MSC group,HGC-27 cell + MSC cell co-culture group and DKK1 + HGC-27 cell + MSC cell co-culture group were induced with osteogenic medium for 16 days and then stained with Alizarin Red.It was found that the addition of DKK1 rescued the loss of osteogenic differentiation caused by HGC-27 cells(p<0.001).Similarly,immunofluorescence showed that DKK1 rescued the decrease in OCN protein expression caused by HGC-27(p<0.001). Western-blot showed OPN and ALP protein expression(p<0.001),ALP activity(p<0.001),and q RT-PCR detection of ALP and OCN m RNA levels(p<0.05)showed partial recovery.The above experiments all proved that DKK1 partially reduced the ability of HGC-27 cells to inhibit osteogenic differentiation of MSCs.These results indicated that the osteoblast differentiation was inhibited before the occurrence of bone metastasis in gastric cancer.Under the influence of HGC-27 cells,the formation of primary cilia in MSCs increases,which was related to the abnormal activation of the Wnt/β-catenin pathway.DKK1 inhibited the Wnt/β-catenin signaling pathway and weakened the degree of HGC-27 cells inhibiting the osteoblast differentiation of MSCs.In summary,our results indicated that gastric cancer cells might activate the Wnt/β-catenin signaling pathway through cilia-dependent activation,causing bone loss before bone metastasis occurs.